1. Technical Field
The present invention generally relates to array based assays and specifically to microarrays for identifying, quantifying, and qualifying target molecules.
2. Background Art
In determining the expression or function of any molecule, traditional methods in molecular biology are only useful at examining the effect of one agent on one cellular molecule, in one experiment, which means that examining any effect on any given molecule, in general, is slow, expensive and difficult to assess. The advent of microarray technology has allowed scientists to examine the effect of one treatment, condition, or combinations thereof on thousands of molecules simultaneously.
Generally, microarray technology consists of probe molecules being attached to a solid substrate and target molecules obtained from the exposed cells contacting the probe molecules. Typically, target molecules are labeled prior to exposure to the microarray. Once exposed to the microarray, some target molecules selectively form probe/target pairs by binding/hybridizing with the complimentary probe molecules on the microarray. The target molecules that do not form pairs are removed from the microarray. Where the probe/target pairs are formed on the microarray, the scientist can then visualize the probe molecules, which were bound by labeled target molecules. The relative amount of probe/target pairs that form can be compared between groups of cells, which are exposed to different treatments and cells that are not (controls) to determine the effect of the treatment. For example, the levels of expression of mRNA or protein as a target molecule may have changed, or alternatively, the conformation of a protein or carbohydrate could have changed. As thousands of molecules can be screened simultaneously using this technology, microarrays can be used to improve timeliness, effectiveness, accuracy and overall benefit-to-cost ratio for examining changes in molecular expression and function relative to traditional methods.
Biochemical molecules on microarrays have been synthesized directly at or on a particular cell (sector) on the microarray, or preformed molecules have been attached to particular cells (sectors) of the microarray by chemical coupling, adsorption or other means. The number of different cells (sectors) and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands. Commercial microarray plate readers typically measure fluorescence in each cell (sector) and can provide data on thousands of reactions simultaneously thereby saving time and labor. A representative example of a patent in the field is U.S. Pat. No. 5,545,531.
Currently, two dimensional arrays of macromolecules are made either by depositing small aliquots on flat surfaces under conditions which allow the macromolecules to bind or be bound to the surface, or the macromolecules can be synthesized on the surface using light-activated or other synthetic reactions. Previous methods also include using printing techniques to produce such arrays. Some methods for producing arrays have been described in “Gene-Expression Micro-Arrays: A New Tool for Genomics”, Shalon, D., in Functional Genomics; Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. & Savage, L. M., eds., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.3.1.-2.3.8; “DNA Probe Arrays: Accessing Genetic Diversity”, Lipshutz, R. J., in Gilbert, S. R. & Savage, L. M., supra, pp 2.4.1.-2.4.16; “Applications of High-Throughput Cloning of Secreted Proteins and High-Density Oligonucleotide Arrays to Functional Genomics”, Langer-Safer, P. R., in Gilbert, S. R. & Savage, L. M., supra; Jordan, B. R., “Large-scale expression measurement by hybridization methods: from high-densities to “DNA chips”, J. Biochem. (Tokyo) 124: 251-8, 1998; Hacia, J. G., Brody, L. C. & Collins, F. S., “Applications of DNA chips for genomic analysis”, Mol. Psychiatry 3: 483-92, 1998; and Southern, E. M., “DNA chips: Analyzing sequence by hybridization to oligonucleotides on a large scale”, Trends in Genetics 12: 110-5, 1996.
One difficulty in microarray technology has been the ability of scientists to efficiently and effectively identify and quantify the probe/target pairs which form on the microarray. As above, the target molecule is typically labeled and that label is detected to identify the probe/target pair. However, the label may not be present on the target molecule in sufficient amounts to be detectable. If a target sequence is not adequately labeled, false negative results are obtained, meaning that probe/target pairs are formed, but not identified by the scientist. Labeling inadequacies often occur due to enzymatic reproducibility, inhibition and or incomplete incorporation of dyes.
Recently, protein and antibody microarrays have been developed using a robotic system used for DNA microarrays (Haab, et al., 2001, Leukling, et al., 1999, MacBeath and Schreiber, 2000, Streekumar, et al., 2001, and Miller, et al., 2003). Scanners and software required to visualize and quantitate fluorescence signals and bioinformatic tools for data mining of DNA arrays can be used for antibody microarray analysis. Moreover, data mining of antibody microarray analyses are almost identical to the DNA arrays. However, development of functional, focused (non-global) antibody arrays is challenging because of a limited number of antibodies that are available for microarray chip production and form-specific antibodies of interest differ greatly in their binding strength to the antigen.
The Lueking et al. (1999) reference disclosed the spotting of 92 crude cell lysates obtained from E. coli transfected with human fetal brain cDNA-containing vectors on the polyvinylidine difluoride (PVDF) membrane. The spotted proteins were hybridized with monoclonal antibodies followed by hybridization with secondary antibodies conjugated with horseradish peroxidase (HRP). Peroxidase activity of the secondary antibody conjugates was detected by incubation of the membrane with CN/DAB solution (Pierce). Meanwhile, the MacBeath and Schreiber (2000) reference disclosed production of microarray slides by conjugating proteins with BSA-N-hydroxysuccinimide coated on glass slides. Using the technology, arrays for three pairs of proteins that were known to interact, three pairs of kinase-substrate proteins, and three pairs of small molecules-binding proteins were produced and the utility of the arrays was explored.
The Haab et al. (2001) reference disclosed production of both protein and antibody microarrays with 115 antigen and antibody (IgG) pairs. Antigen proteins or antibodies were spotted on poly-L-lysine coated glass with a cross-linking layer. Concentration of the antigen proteins or antibodies spotted on the slides was limited to any single concentration between 0.1 and 0.3 mg/ml. Contrary to the single concentration of the antigen proteins or antibodies spotted on the slide, six different concentrations of antigen proteins or antibodies were conjugated with dyes and hybridized with the spotted antigen proteins or antibodies to test sensitivity. It was found that 50% of the arrayed antigen proteins and 20% of the arrayed antibodies provided meaningful data at or below concentrations of 0.34 μg/ml and 1.6 μg/ml, respectively.
The Sreekumar et al. (2001) reference disclosed the production of antibody microarrays with poly-L-lysine coated or superaldehyde-modified glass slides (Telechem International Inc., Sunnyvale, Calif.). This was a targeted microarray of 146 antibodies for stress response, cell cycle progression, and apoptosis spotted with single concentration of antibodies.
Finally, the Miller et al. (2003) reference disclosed the spotting of 184 monoclonal and polyclonal immunoglobulin (IgG) on poly-L-lysine (CEL Associates, Pearland, Tex.) with a photoreactive cross-linking layer (Molecular Biosciences, Boulder, Colo.) or polyacrylamide-based hydrogel (Packard Bioscience, Meriden, Conn.) glass slides. Concentration of the antibodies spotted on the slide was limited to any single concentration between 0.1 and 0.3 mg/ml. This was a targeted antibody array used for serum prostate cancer marker screening. Thus, 40 antibodies for serum proteins detected in the serum of normal person and 13 antibodies for proteins detected in the serum of cancer patients were included.
In view of the prior art, no antibody microarrays have been developed for anti-sera or ascites fluids. Accordingly, the present invention relates to spotting increased levels of anti-sera or ascites fluids to compensate for proteins other than IgG in the anti-sera or ascites fluids, which made it possible to include additional form-specific antibodies. Contrary to DNA microarrays produced to detect global gene expression, it is impossible to produce antibody microarrays to detect global protein expression primarily due to small number (lower than 500) of antibodies spotted on the chip. Thus, bias of data resulted from the unique set of the antibodies spotted on the arrays has to be corrected with a spiked internal control, which is not expressed in the samples. Use of the internal control for normalization of the target arrays has not been previously reported. So far, single concentration of IgG has been spotted for array analysis. In the present invention, a method to determine optimal IgG levels to obtain optimal signal levels in microarrays by spotting various concentrations of IgG is disclosed. In the present invention, the advantage of spotting various concentrations of IgG is also disclosed.
Drug metabolizing enzyme antibody microarrays were produced using purified immunoglobulins (IgG) as well as fluids unprocessed for IgG isolation, e.g., anti-sera or ascites fluids. They were used to analyze protein expression of hepatic proteins obtained after phenobarbital treatment of rats. Twelve up-regulated proteins after phenobarbital treatment were identified by the antibody microarray. It was surprising that, in Western blot analysis, only 1 out of the 12 up-regulated proteins failed to show increased protein expression.